Generation of nano-crystals by microbes, both intracellularly and extracellularly, has been reported for quite sometime now. Reductases and quinones are known to facilitate this process. But little is known on the role of the microbes in enhancing the size of the crystal formed. Turbidometric method for determination of soluble sulfate concentration was used for the generation of the barium sulfate crystals both from purely chemical as well as biological (cell free supernatant) means. Transmission Electron Microscopy (TEM) was used for visualization of the stained crystal followed by measurement of the crystal size. Statistical analysis of the variation in size was done using a two-sample one-tailed Z-test. Under identical conditions of generation, the dimensions of the crystals from biological origin were much enlarged [363.54 nm (horizontal), 399.89 nm (vertical)] compared to those from purely chemical origin [265.54 nm (horizontal), 286.46 nm (vertical)]. The Z-test and computation of the corresponding p-value indicated that the variation was highly significant.

Barites whose chemical composition is Barium sulfate crystals, are commonly
used for commercial applications like filler for thermosetting and thermostable
plastics (to increase hardness and rigidity without disturbing the toughness
and surface quality), elastomers, sealants, adhesives, varnishes, paints, paper,
glass and as substrate for colored pigment formulations as well as for single-layer
or multilayer coatings consisting of metal oxides, metal oxide mixtures and/or
metal compounds (US Patent 7501110). They are also suitable for producing semi-opaque
colourings for lamp coverings (US Patent 7501110). Medical applications include
their use as X-ray contrast agent for examination of the gastrointestinal tract
(US Patent 7501110; USPC 423170). It results in a distinctly higher X-ray opacity
for medical articles. The particle size can be influenced by varying the concentration,
temperature and stirring speed (Gardner and Nancollas, 1983).

Soluble sulfate load in effluent water is a major industrial problem for the
mining sector which calls for immediate attention. Though chemical methods are
available but more emphasis has been put on the application of microbe based
bioremediation (Nasipuri et al., 2010). The measurement
of sulfate concentration is done using turbidometric technique, (Nasipuri
et al., 2010; Icgen et al., 2006)
which results in formation of barium sulfate crystals. This study looks into
the effect of the cell free supernatant from sulfate reducing bacterial consortium
(isolated from East Calcutta Wetland) on the crystal size enhancement when compared
to the same generated using pure chemical solutions. We ascribe the enhancement
of crystal size to possible extracellular reductases.

Generation of barium sulfate crystals: The turbidometric method was
used for barium sulfate crystal generation from cell free supernatant of the
SRB consortium (Nasipuri et al., 2010; Icgen
et al., 2006). The control sample was prepared by similarly treating
sodium sulfate solution with one gram of barium chloride crystals. In both the
cases the suspended crystals were directly used for transmission electron microscopy.

Transmission electron microscopy: Formvar coated copper grids were used for immobilization of the crystals from 20 μL of the suspension mentioned above. The grids were placed on the suspension drop for two minutes. Then the grid was placed over a drop of 0.2% uranyl acetate for 2 min. The excess stain was washed off carefully in three consecutive drops of filtered sterile milli Q water. The excess water was soaked with a blotting paper and then the grid was kept for drying overnight in a desiccator. The grids were visualized at 14000X magnification with 60 kV acceleration voltage in a Jeol JEM 100S. The images were photographed from the fluorescent screen using digital camera (Samsung S860) and printed on papers. Each crystal image was measured using vernier caliper. The measurement was carried out in both horizontal and vertical directions. The final crystal dimension was calculated taking into account the magnification of the image. Dimensions of 125 crystals were measured in both the cases.

Statistical analysis: The objective of the study was to investigate whether there was any significant difference between the dimensions (in both horizontal and vertical directions) of the crystals prepared by chemical means and of those coming from biological origin, i.e., cell free extract. In both cases (control, i.e. prepared by chemical means and sample, i.e., created from cell free extract), 125 crystals each were used for statistical analysis of the data.

The null and alternative hypotheses can be formulated as follows in case of
the horizontal dimension:

H0

:

The dimension (horizontal) of the crystals originating from
chemical and biological means are equal, i.e., μ1 = μ2

H1

:

The dimension (horizontal) of the crystals originating from biological
means is greater than that originating from chemical means, i.e., μ2>μ1

Sample size n = 125

α = 5%

Since, the sample size was large, a two-sample one-tailed Z-test was performed using the following formula:

(1)

Where:

=

Sample mean of the horizontal dimensions in case of crystals
from chemical origin

=

Sample mean of the horizontal dimensions in case of crystals from biological
origin

S12

=

Sample variance of the horizontal dimensions in case of crystals from
chemical origin

S22

=

Sample variance of the horizontal dimensions in case of crystals from
biological origin

n1

=

Sample size for chemically prepared crystals

n2

=

Sample size for biologically prepared crystals

An exactly similar set of hypotheses was set up in case of the vertical dimension using the same sample size and level of significance.

Project details: Project was sponsored by Department of Atomic Energy, Government of India under the BRNS scheme with Grant No. 2005/36/31/BRNS/2574 dated 2nd March 2006. The fellowship was from Council of Scientific and Industrial Research with Grant No. 09/933(0001)/2006-EMR-I, India and Department of Biotechnology with Grant No. BT/HRD/01/015/2000 dated 13/8/2002, Government of India.

RESULTS AND DISCUSSION

A summary of the data obtained from measurements on the two types of crystals
(Fig. 1a, b), viz., control (i.e., chemical)
and sample (i.e., biological) is given in Table 1.

The result from the two-sample one-tailed test of hypothesis is shown below:

Horizontal:

Zcalculated

=

-10.39 (the critical value of z at 5% level of significance
is 1.64)

p-value

=

0

Fig. 1:

Transmission electron micrograph of BaSO4 crystals
at 14000X magnification. BaSO4 crystals were formed according
to the turbidimetric method. Crystals were immobilized on formvar coated
300 mesh copper grids from Pro Sci. Tech. (GSCu300C-50) and negatively stained
with 0.2% Uranyl acetate solution for 2 min. The excess stain was washed
off carefully in three consecutive drops of filtered sterile milliQ water
and air dried. The sample was visualized under TEM. Jeol (JEM 100S), (a)
BaSO4 crystals formed from Na2SO4 solution
upon addition of 1 g of BaCl2 under acidic condition according
to the turbidometric method. The dimensions of the crystal are given in
cm and (b) BaSO4 crystals formed from cell free supernatant containing
soluble sulfate according to turbidometric method. The dimensions of the
crystal are given in cm

Table 1:

Tabular representation of the mean and variance of the crystal
dimensions generated through chemical (control) and biological (sample)
means

The low p-values clearly show that there is enough statistical evidence to
reject the null hypotheses. This is corroborated by the fact that values of
z calculated on the basis of the observed data fall in the critical region of
the normal distributions. It means that the two sets (crystals obtained from
chemical and biological sources) are drawn from populations which have different
mean values. In other words, the dimensions (horizontal as well as vertical)
of the crystals of chemical and biological origins are significantly different
from one another. This enhancement in crystal dimension could be due to the
extracellular enzymes secreted by the microbial consortia. This consortium also
revealed the presence of Dissimilatory Sulfite Reductase A as reported elsewhere
(Nasipuri et al., 2010). That could be the putative
reason for the enlargement phenomenon observed in case of the samples prepared
by biological means keeping the other conditions identical. Here, we report
for the first time the crystal dimension enhancement property of SRB consortium
predominated by Desulfovibrio sp. Reductases from biological origin (yeast,
fungus and bacteria like nitrate reducers and Lactobacillus) (Duran
et al., 2005; Ahmad et al., 2003;
Kumar et al., 2007; Vaidyanathan
et al., 2010) have been reported to perform the function of crystal
generation, however this study focuses on the crystal size enhancement ability
of the extracellular microbial enzymes. The future extension of this study would
be to fine tune the reaction to the point of fabricating barite crystals of
predetermined size. An analysis of the extracellular supernatant for detection
of reductase activity by biochemical means and characterization of the purified
enzyme would help in understanding the process clearly.

ACKNOWLEDGMENTS

The authors would like to acknowledge the fruitful discussion with Prof A.K. Rakshit during preparation of the study. Project was sponsored by Department of Atomic Energy, Government of India under the BRNS scheme with Grant No. 2005/36/31/BRNS/2574 dated 2nd March 2006. The fellowship was from Council of Scientific and Industrial Research with Grant No. 09/933(0001)/2006-EMR-I, India and Department of Biotechnology with Grant No. BT/HRD/01/015/2000 dated 13/8/2002, Government of India.